Identification of novel factors involved in the exacerbation of HIV-1 infection and spread among macrophages in the tuberculosis context

Mycobacterium tuberculosis (Mtb), la bactérie responsable de la tuberculose (TB), et le virus de l'immunodéficience humaine (VIH-1), l'agent du syndrome de l'immunodéficience acquise (SIDA), accélèrent leurs progressions mutuelles chez les patients co-infectés. Alors que de nombreuses données cliniques rapportent une augmentation de la charge virale dans les sites anatomiques co-infectés, les mécanismes qui en sont responsables restent insuffisamment décrits. Mtb cible principalement les macrophages. Nous émettons l'hypothèse que l'infection des macrophages par Mtb créé un microenvironnement propice à la réplication du VIH-1 au niveau des sites co-infectés. Pour le montrer, j'ai utilisé un modèle in vitro précédemment établi par mes équipes (le cmMTB - pour " conditioned media of Mtb-infected macrophages "). Celui-ci permet de mimer un environnement tuberculeux, par la différenciation et l'activation des macrophages vers un profil " M(cmMTB) ", largement retrouvé dans les poumons lors d'une tuberculose active. En rejoignant le laboratoire, j'ai participé à l'étude des mécanismes responsables de l'augmentation de la réplication virale dans le contexte de co-infection, en utilisant ce modèle. Nous avons trouvé que les M(cmMTB) forment de nombreux nanotubes (ponts intercellulaires), leur permettant de transférer plus de virus d'un macrophage à l'autre, et conduit à une forte augmentation de la production virale. L'objectif principal de ma thèse a donc été d'identifier, dans un contexte tuberculeux, de nouveaux facteurs impliqués dans l'augmentation de la réplication du VIH-1 dans les macrophages. Pour cela, une analyse transcriptomique des M(cmMTB) a été réalisée, révélant deux facteurs essentiels : le récepteur Siglec-1 et les interférons de type I (IFN-I) via STAT1. Dans un premier temps, j'ai étudié le rôle de Siglec-1 dans la synergie entre Mtb et le VIH-1 dans les macrophages. D'abord, j'ai montré que son expression de surface était augmentée par le cmMTB, de façon dépendante des IFN-I. Ensuite, j'ai établi que l'abondance des macrophages alvéolaires exprimant Siglec-1 chez les primates non-humains co-infectés avec Mtb et le virus de l'immunodéficience simienne corrélait avec la sévérité de la pathologie, et était associée à la signalisation des IFN-I, via l'activation de STAT1. De plus, j'ai identifié une nouvelle localisation de Siglec-1 le long d'un sous-type de nanotubes. Ceux-ci, plus larges et plus longs, contenaient plus de VIH-1 que les autres, suggérant que le virus puisse les emprunter en majorité. Enfin, j'ai montré que Siglec-1 était responsable de l'augmentation de la réplication du VIH-1, grâce à une meilleure capture du virus et de son transfert intercellulaire, potentiellement grâce aux nanotubes. Ces résultats permettent de proposer que Siglec-1 a un rôle physiologique dans la biologie des nanotubes et des macrophages. Dans un second temps, j'ai déterminé que la TB dérégule la signalisation IFN-I/STAT1 dans les macrophages et diminue leur réponse antivirale. Après stimulation des M(cmMTB) par de l'IFN-I exogène, j'ai démontré une diminution de l'activation de STAT1 et de l'expression des gènes de réponse à l'IFN, indiquant une potentielle désensibilisation de ces cellules à l'IFN-I. Ces observations, ainsi que la perte de contrôle de la réplication du VIH-1, ont été reversées par l'inhibition du récepteur aux IFN-I (IFNAR2) ou du facteur de transcription STAT1. Ensemble, ces résultats indiquent que les IFN-I ont un rôle délétère pour l'hôte dans le contexte de la co-infection. Mon projet de thèse a permis de révéler la capacité de Mtb à déréguler les réponses antivirales de l'hôte contre le VIH-1, participant ainsi à la gravité de la co-infection. Les facteurs identifiés au cours de ma thèse pourraient à plus long terme être utilisés à des fins diagnostiques ou thérapeutiques, dans le but d'améliorer la prise en charge des patients co-infectés.Mycobacterium tuberculosis (Mtb), the bacteria causing tuberculosis (TB), and the human immunodeficiency virus type 1 (HIV-1), the etiological agent of acquired immunodeficiency syndrome (AIDS), act in synergy to exacerbate the progression of each other in co-infected patients. While clinical evidence reveals a frequent increase of the viral load at co-infected anatomical sites, the mechanisms explaining how Mtb favours HIV-1 progression remain insufficiently understood. Macrophages are the main target for Mtb. Their infection by the bacilli likely shapes the microenvironment that favours HIV-1 infection and replication at sites of co-infection. To address this issue, I took advantage of an in vitro model mimicking the TB-associated microenvironment (cmMTB, "conditioned media of Mtb-infected macrophages") previously established in the laboratory; a model that renders macrophages susceptible to intracellular pathogens like Mtb. Upon joining the team, I participated in the study on how Mtb exacerbates HIV-1 replication in macrophages, using this model. We found that cmMTB-treated macrophages (M(cmMTB)) have an enhanced ability to form intercellular membrane bridges called tunneling nanotubes (TNT), which increase the capacity of the virus to transfer from one macrophage to another, leading to the exacerbation of HIV-1 production and spread. The principal objective of my PhD thesis was to identify novel factors that are involved in the exacerbation of HIV-1 replication in macrophages in the context of tuberculosis. To this end, a transcriptomic analysis of M(cmMTB) was conducted, and revealed two key factors: the Siglec-1 receptor and type I interferon (IFN-I)/STAT1 signaling. The first part of my PhD thesis dealt with the characterization of Siglec-1 as a novel factor involved in the synergy between Mtb and HIV-1 in macrophages. First, I demonstrated that its increased expression in M(cmMTB) was dependent on IFN-I. Second, in Mtb and simian immunodeficiency virus co-infected non-human primates, I established a positive correlation between the abundance of Siglec-1+ alveolar macrophages and the pathology, associated with the activation of the IFN-I/STAT-1 pathway. Third, I revealed that Siglec-1 is localized on a specific subset of thick TNT. These thick Siglec-1+ TNT were longer and contained high HIV-1 cargos compared to Siglec-1- TNT, suggesting that the virus might preferentially use these TNT. Finally, I showed that Siglec-1 was responsible for the enhanced HIV-1 replication by increasing viral capture and cell-to-cell transfer, at least in part through TNT. These findings argue that Siglec-1 has a physiological significance to macrophage and TNT biology. It may also be a potential target for designing new therapies aimed at reducing viral dissemination in a co-infection context. In the second part of my PhD project, I determined that TB dysregulates the IFN-I/STAT-1 signaling pathway to diminish the antiviral response. Indeed, upon exogenous IFN-I stimulation, I noticed a reduced STAT1 activation and lower expression of the interferon-stimulated genes (ISG) in M(cmMTB) compared to control cells, indicating a potential desensitization to the IFN-I/STAT-1 pathway. By blocking IFNAR2 or inhibiting STAT-1 gene expression during cmMTB-treatment, I prevented this desensitization to exogenous IFN-I and restored control of HIV-1 infection in macrophages. Altogether, these results point to a deleterious role of IFN-I in the co-infection setting. My thesis work unveils the capacity of Mtb to dysregulate host-antiviral responses against HIV-1. Modulation of the macrophage response by TB-induced IFN-I, including upregulation of Siglec-1 expression, explains how these cells become vessels for viral spread that contributes to co-infection severity. In addition, the identified factors in this project may be used as new diagnostic tools or therapeutic targets to ameliorate the diagnosis and treatment of co-infected individuals

Density dependent regulation of inflammatory responses in macrophages

Macrophage distribution density is tightly regulated within the body, yet the importance of macrophage crowding during in vitro culture is largely unstudied. Using a human induced pluripotent stem cell (iPSC)-derived macrophage model of tissue resident macrophages, we characterize how increasing macrophage culture density changes their morphology and phenotype before and after inflammatory stimulation. In particular, density drives changes in macrophage inflammatory cytokine and chemokine secretion in both resting and activated states. This density regulated inflammatory state is also evident in blood monocyte derived-macrophages, the human monocytic THP-1 immortalized cell line, and iPSC-derived microglia. Density-dependent changes appear to be driven by a transferable soluble factor, yet the precise mechanism remains unknown. Our findings highlight cell plating density as an important but frequently overlooked consideration of in vitro macrophage research relevant to a variety of fields ranging from basic macrophage cell biology to disease studies

Tuberculosis Exacerbates HIV-1 Infection through IL-10/STAT3-Dependent Tunneling Nanotube Formation in Macrophages

The tuberculosis (TB) bacillus, Mycobacterium tuberculosis (Mtb), and HIV-1 act synergistically; however, the mechanisms by which Mtb exacerbates HIV-1 pathogenesis are not well known. Using in vitro and ex vivo cell culture systems, we show that human M(IL-10) anti-inflammatory macrophages, present in TB-associated microenvironment, produce high levels of HIV-1. In vivo, M(IL-10) macrophages are expanded in lungs of co-infected non-human primates, which correlates with disease severity. Furthermore, HIV-1/Mtb co-infected patients display an accumulation of M(IL-10) macrophage markers (soluble CD163 and MerTK). These M(IL-10) macrophages form direct cell-to-cell bridges, which we identified as tunneling nanotubes (TNTs) involved in viral transfer. TNT formation requires the IL-10/STAT3 signaling pathway, and targeted inhibition of TNTs substantially reduces the enhancement of HIV-1 cell-to-cell transfer and overproduction in M(IL-10) macrophages. Our study reveals that TNTs facilitate viral transfer and amplification, thereby promoting TNT formation as a mechanism to be explored in TB/AIDS potential therapeutics

Dissemination of <i>Mycobacterium tuberculosis</i> is associated to a <i>SIGLEC1</i> null variant that limits antigen exchange via trafficking extracellular vesicles

The identification of individuals with null alleles enables studying how the loss of gene function affects infection. We previously described a non‐functional variant in SIGLEC1, which encodes the myeloid‐cell receptor Siglec‐1/CD169 implicated in HIV‐1 cell‐to‐cell transmission. Here we report a significant association between the SIGLEC1 null variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. Local spread of bacteria within the lung is apparent in Mtb‐infected Siglec‐1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. We find that Siglec‐1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec‐1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination

A rapid antibody screening haemagglutination test for predicting immunity to SARS-CoV-2 variants of concern

Background: Evaluation of susceptibility to emerging SARS-CoV-2 variants of concern (VOC) requires rapid screening tests for neutralising antibodies which provide protection. Methods: Firstly, we developed a receptor-binding domain-specific haemagglutination test (HAT) to Wuhan and VOC (alpha, beta, gamma and delta) and compared to pseudotype, microneutralisation and virus neutralisation assays in 835 convalescent sera. Secondly, we investigated the antibody response using the HAT after two doses of mRNA (BNT162b2) vaccination. Sera were collected at baseline, three weeks after the first and second vaccinations from older (80–99 years, n = 89) and younger adults (23–77 years, n = 310) and compared to convalescent sera from naturally infected individuals (1–89 years, n = 307). Results: Here we show that HAT antibodies highly correlated with neutralising antibodies (R = 0.72–0.88) in convalescent sera. Home-dwelling older individuals have significantly lower antibodies to the Wuhan strain after one and two doses of BNT162b2 vaccine than younger adult vaccinees and naturally infected individuals. Moverover, a second vaccine dose boosts and broadens the antibody repertoire to VOC in naïve, not previously infected older and younger adults. Most (72–76%) older adults respond after two vaccinations to alpha and delta, but only 58–62% to beta and gamma, compared to 96–97% of younger vaccinees and 68–76% of infected individuals. Previously infected older individuals have, similarly to younger adults, high antibody titres after one vaccination. Conclusions: Overall, HAT provides a surrogate marker for neutralising antibodies, which can be used as a simple inexpensive, rapid test. HAT can be rapidly adaptable to emerging VOC for large-scale evaluation of potentially decreasing vaccine effectiveness.publishedVersio

Identification de nouveaux facteurs impliqués dans l’augmentation de l’infection et la dissémination du VIH-1 dans un contexte de Tuberculose

Mycobacterium tuberculosis (Mtb), the bacteria causing tuberculosis (TB), and the human immunodeficiency virus type 1 (HIV-1), the etiological agent of acquired immunodeficiency syndrome (AIDS), act in synergy to exacerbate the progression of each other in co-infected patients. While clinical evidence reveals a frequent increase of the viral load at co-infected anatomical sites, the mechanisms explaining how Mtb favours HIV-1 progression remain insufficiently understood. Macrophages are the main target for Mtb. Their infection by the bacilli likely shapes the microenvironment that favours HIV-1 infection and replication at sites of co-infection. To address this issue, I took advantage of an in vitro model mimicking the TB-associated microenvironment (cmMTB, "conditioned media of Mtb-infected macrophages") previously established in the laboratory; a model that renders macrophages susceptible to intracellular pathogens like Mtb. Upon joining the team, I participated in the study on how Mtb exacerbates HIV-1 replication in macrophages, using this model. We found that cmMTB-treated macrophages (M(cmMTB)) have an enhanced ability to form intercellular membrane bridges called tunneling nanotubes (TNT), which increase the capacity of the virus to transfer from one macrophage to another, leading to the exacerbation of HIV-1 production and spread. The principal objective of my PhD thesis was to identify novel factors that are involved in the exacerbation of HIV-1 replication in macrophages in the context of tuberculosis. To this end, a transcriptomic analysis of M(cmMTB) was conducted, and revealed two key factors: the Siglec-1 receptor and type I interferon (IFN-I)/STAT1 signaling. The first part of my PhD thesis dealt with the characterization of Siglec-1 as a novel factor involved in the synergy between Mtb and HIV-1 in macrophages. First, I demonstrated that its increased expression in M(cmMTB) was dependent on IFN-I. Second, in Mtb and simian immunodeficiency virus co-infected non-human primates, I established a positive correlation between the abundance of Siglec-1+ alveolar macrophages and the pathology, associated with the activation of the IFN-I/STAT-1 pathway. [...]Mycobacterium tuberculosis (Mtb), la bactérie responsable de la tuberculose (TB), et le virus de l'immunodéficience humaine (VIH-1), l'agent du syndrome de l'immunodéficience acquise (SIDA), accélèrent leurs progressions mutuelles chez les patients co-infectés. Alors que de nombreuses données cliniques rapportent une augmentation de la charge virale dans les sites anatomiques co-infectés, les mécanismes qui en sont responsables restent insuffisamment décrits. Mtb cible principalement les macrophages. Nous émettons l'hypothèse que l'infection des macrophages par Mtb créé un microenvironnement propice à la réplication du VIH-1 au niveau des sites co-infectés. Pour le montrer, j'ai utilisé un modèle in vitro précédemment établi par mes équipes (le cmMTB - pour " conditioned media of Mtb-infected macrophages "). Celui-ci permet de mimer un environnement tuberculeux, par la différenciation et l'activation des macrophages vers un profil " M(cmMTB) ", largement retrouvé dans les poumons lors d'une tuberculose active. En rejoignant le laboratoire, j'ai participé à l'étude des mécanismes responsables de l'augmentation de la réplication virale dans le contexte de co-infection, en utilisant ce modèle. Nous avons trouvé que les M(cmMTB) forment de nombreux nanotubes (ponts intercellulaires), leur permettant de transférer plus de virus d'un macrophage à l'autre, et conduit à une forte augmentation de la production virale. L'objectif principal de ma thèse a donc été d'identifier, dans un contexte tuberculeux, de nouveaux facteurs impliqués dans l'augmentation de la réplication du VIH-1 dans les macrophages. Pour cela, une analyse transcriptomique des M(cmMTB) a été réalisée, révélant deux facteurs essentiels : le récepteur Siglec-1 et les interférons de type I (IFN-I) via STAT1. Dans un premier temps, j'ai étudié le rôle de Siglec-1 dans la synergie entre Mtb et le VIH-1 dans les macrophages. D'abord, j'ai montré que son expression de surface était augmentée par le cmMTB, de façon dépendante des IFN-I. Ensuite, j'ai établi que l'abondance des macrophages alvéolaires exprimant Siglec-1 chez les primates non-humains co-infectés avec Mtb et le virus de l'immunodéficience simienne corrélait avec la sévérité de la pathologie, et était associée à la signalisation des IFN-I, via l'activation de STAT1. De plus, j'ai identifié une nouvelle localisation de Siglec-1 le long d'un sous-type de nanotubes.[...

Tunneling Nanotubes: Intimate Communication between Myeloid Cells

Tunneling nanotubes (TNT) are dynamic connections between cells, which represent a novel route for cell-to-cell communication. A growing body of evidence points TNT towards a role for intercellular exchanges of signals, molecules, organelles, and pathogens, involving them in a diverse array of functions. TNT form among several cell types, including neuronal cells, epithelial cells, and almost all immune cells. In myeloid cells (e.g., macrophages, dendritic cells, and osteoclasts), intercellular communication via TNT contributes to their differentiation and immune functions. Importantly, TNT enable myeloid cells to communicate with a targeted neighboring or distant cell, as well as with other cell types, therefore creating a complex variety of cellular exchanges. TNT also contribute to pathogen spread as they serve as “corridors” from a cell to another. Herein, we addressed the complexity of the definition and in vitro characterization of TNT in innate immune cells, the different processes involved in their formation, and their relevance in vivo. We also assess our current understanding of how TNT participate in immune surveillance and the spread of pathogens, with a particular interest for HIV-1. Overall, despite recent progress in this growing research field, we highlight that further investigation is needed to better unveil the role of TNT in both physiological and pathological conditions

Tunneling Nanotubes: Intimate Communication between Myeloid Cells

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